Near‐Infrared‐II Nanoparticles for Vascular Normalization Combined with Immune Checkpoint Blockade via Photodynamic Immunotherapy Inhibit Uveal Melanoma Growth and Metastasis

Abstract Photodynamic therapy (PDT) has been widely employed in tumor treatment due to its effectiveness. However, the tumor hypoxic microenvironment which is caused by abnormal vasculature severely limits the efficacy of PDT. Furthermore, the abnormal vasculature has been implicated in the failure of immunotherapy. In this study, a novel nanoparticle denoted as Combo‐NP is introduced, composed of a biodegradable NIR II fluorescent pseudo‐conjugate polymer featuring disulfide bonds within its main chain, designated as TPA‐BD, and the vascular inhibitor Lenvatinib. Combo‐NP exhibits dual functionality by not only inducing cytotoxic reactive oxygen species (ROS) to directly eliminate tumor cells but also eliciting immunogenic cell death (ICD). This ICD response, in turn, initiates a robust cascade of immune reactions, thereby augmenting the generation of cytotoxic T lymphocytes (CTLs). In addition, Combo‐NP addresses the issue of tumor hypoxia by normalizing the tumor vasculature. This normalization process enhances the efficacy of PDT while concurrently fostering increased CTLs infiltration within the tumor microenvironment. These synergistic effects synergize to potentiate the photodynamic‐immunotherapeutic properties of the nanoparticles. Furthermore, when combined with anti‐programmed death‐ligand 1 (PD‐L1), they showcase notable inhibitory effects on tumor metastasis. The findings in this study introduce an innovative nanomedicine strategy aimed at triggering systemic anti‐tumor immune responses for the treatment of Uveal melanoma.


General measurements
1 H NMR spectra was measured by a 300 MHz and 400 MHz NMR spectrometer (Bruker, Germany) at room temperature.The morphology and size of nanoparticles were measured by transmission electron microscope (TEM) carried out with a HT7700 electron microscope.Size and zeta potential measurements were conducted via a Malvern Zetasizer (Nano ZS, UK).The absorption spectrum was measured using an ultraviolet-visible spectrometer (UV-vis, UV-2600, Shimadzu, Japan).Immunofluorescence images were conducted using a confocal laser scanning microscope (CLSM, FV1000-IX8l, Olympus, Japan).The MTT assay was conducted using a Microplate reader (SpectraMax M3).The mice imaging was conducted by an In Vivo Imaging System (IVIS, PerkinElmer).Flow cytometry (FCM) was done by a flow cytometry analyzer (Beckman Coulter, USA).
Next, the NEt3 (15 mL) was added and stirred at room temperature for another 15 min.Then, the boron (III) fluoride ethylether complex (75 mL) was added and stirred at room temperature for 3 h.When TLC analysis indicated that the reaction was complete, the mixture was diluted with DCM (100 mL × 3), washed with water (200 mL) and saturated NaCl solution (100 mL).
The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo.

Synthesis of compound 3
The compound 3 was synthesized as previously described. [2]To a solution of monomer 2 (100.0 g, 0.15 mmol) and 4-bromobenzaldehyde (83.3 mg, 0.45 mmol) in toluene (10 mL) was added beta-alanine (10 mg) and piperidine (20 μL).The mixture was stirred at 80 ℃ for 24 h.Then, the solvent was evaporated to afford the crude product, which was purified by silica gel column chromatography to give compound 3 as a dark green solid (84.0 mg, 86.4% yield).
The polymerization reaction was carried out at 120 ℃ under nitrogen atmosphere in dark for and then purified by sedimentation in methanol.The black solid of TPA-BD was obtained.

Formulation of Len-NP, PDT-NP and Combo-NP
Briefly, the TPA-BD polymer (10 mg) was dissolved in THF (5 mL), P1(50 mg) and mDSPE-PEG2000 (50 mg) were completely dissolved in DMSO (3 mL), and then added into 40 mL pure water under continuous sonication.Lenvatinib (10 mg), P1(50 mg) and DSPE-PEG2000 (50 mg) were dissolved in DMSO (4 mL), and then added dropwise into 20 mL pure water with magnetic stirring.Afterward, the organic solvent was cleared by dialysis for 24 hours and then PDT-NP, Len-NP were obtained.The Combo-NP solution was a mixture of PDT-NP and Len-NP in certain ratio.All nanoparticle solutions were stored at 4 °C for further use.

Singlet oxygen generation of PDT-NP
The singlet oxygen ( 1 O2) generation ability by PDT-NP under the 808 nm irradiation was detected using the chemical probe 1,3-diphenylisobenzofuran (DPBF), which reacted irreversibly with 1 O2 to cause a decrease in the DPBF absorption at about 410 nm.Briefly, the absorbance of DPBF at 410 was adjusted to about 1.0, the absorbance of PDT-NP was adjusted to about 0.2.Then the cuvette was irradiated (808 nm, 1 W/cm 2 ).Characteristic absorbance at 410 nm was recorded by Microplate reader (SpectraMax, USA).

Cell culture
OCM1 and B16F10 cells were cultured in DMEM (glucose 4.5 g/L) supplemented with 10% fetal bovine serum and 1% P/S at 37 ℃ with 5% CO2.When cell fusion reached 80%-90%, the cells were digested with 0.25% trypsin, and then passaged and cultured or inoculated into cell plates for subsequent experiments.

Cellular internalization analysis of nanoparticles
Cover slides were placed in the bottom of each well of a 24-well plate.OCM1 cells (1 × 10 5 ) in 1 mL media were added to each well and incubated at 37 °C for overnight.Then the cells were treated with Combo-NP at the concentration of PDT-NP 5 μg/mL for 1 h, 3 h or 6 h respectively.After being washed with cold PBS for three times, the cell was fixed with paraformaldehyde and cell nuclei was stained with DAPI.Subsequently, images were collected by CLSM (DAPI, λex = 405 nm, Combo-NP, λex = 808 nm).The CLSM imaging system was FLIM-confocal-AFM (Multi-dimensional confocal microscopy fluorescence imaging system, light guide, up-conversion), produced by the U.S. ISS company.Furthermore, the internalization was further detected and quantified by FCM.OCM1 cells (20 × 10 5 ) in 1 mL media were added to each well and incubated at 37 °C for overnight.Then the cells were treated with Combo-NP@Cy5.5 for 1 h, 3 h or 6 h respectively.Then washed with PBS and analyzed by FCM.

Cellular ROS generation
Intracellular ROS level was investigated by using DCFH-DA as a fluorescent probe.In brief, cover slides were placed into the bottom of each well of a 24-well plate.OCM1 cells (1 × 10 5 ) in 1 mL media were added to each well and incubated at 37 °C overnight and then treated with Len-NP, PDT-NP and Combo-NP at the same concentration of photosensitizer (10 μg/mL).
After 6 h treatment, cells were washed and then cultured in serum-free medium with DCFH-DA (10 μM) for 20 mins, then the cells of laser groups were irradiated with 808 nm laser.
Afterward, the cells were fixed with paraformaldehyde and cell nuclei were stained with DAPI.
Subsequently, images were collected with CLSM.Furthermore, the intracellular ROS level was further detected and quantified by FCM.Firstly, cells were seeded in 12-well plate at a density of 2 × 10 5 per well and incubated at 37 °C overnight.Afterward, the cells were treated with the same approach and conditions as the above CLSM analysis.Finally, the cells were harvested immediately to examine the intracellular ROS by FCM.

Cytotoxicity assay in vitro
OCM1 and B16F10 cells were seeded in 96-well plates (6 × 10 3 cells per well) and cultured with complete medium (10% FBS) at 37 ℃ for overnight.Then the cells were divided into 6 groups including PBS, Len-NP, PDT-NP, Combo-NP, PDT-NP+L and Combo-NP+L (Lenvatinib and photosensitizer concentrations ranging from 1.3 μM to 20 μM and 0.6 μg/mL to 10 μg/mL, respectively) and incubated for another 12 h.The cells in the laser groups were irradiated with NIR light of 808 nm at power of 1.0 W/cm 2 for 2 min.Finally, the cell viability was determined using MTT assay.For live/dead cell staining of 3D tumor spheroids, 1% agarose gel solution (50 μL) was added to each well.Next, 1500 cells (200 μL complete medium) were added into each well.The cell spheres were basically formed after 7 days, then different formulations were added and incubated another 12 h.The cells of laser groups were irradiated with a NIR light of 808 nm at intensity of 1.0 W/cm 2 for 2 min.Cells were continued to be cultured for 12 h and then washed with PBS.Afterwards, cells were successfully stained with PI base on the manufacturer's instruction of the live/dead cell staining kit, respectively.
Lastly, the cells were imaged by CLSM with Z-stack scanning.

Apoptosis analysis in vitro
Cellular apoptosis was assessed with an Annexin V-FITC apoptosis detection kit according to the manufacture's instruction.OCM1 and B16F10 cells were cultured in 12-well plates (2 × 10 5 cells per well) overnight.Afterwards, the cells were treated with PBS, Len-NP, PDT-NP, Combo-NP, PDT-NP+L and Combo-NP+L (Lenvatinib and photosensitizer concentration of 5 μM and 2.5 μg/mL) for 12 h.The cells in the laser groups were irradiated with NIR light of 808 nm and cultured for another 12 h.Finally, the cells were incubated with Annexin/PI reagent in the dark for 20 min and immediately measured with FCM.

ICD induced by Combo-NP in vitro
To determine different formulations-induced immunogenic cell death (ICD) of the tumor cells, the secretion of adenosine triphosphate (ATP), calreticulin (CRT) exposure, and extracellular release of high mobility group box 1 (HMGB1) were examined in vitro.In order to study the exposed CRT on the cell surface, cover slides were placed into the bottom of each well of a 24well plate.The cells (1 × 10 5 ) in 1 mL media were added to each well and incubated at 37 °C overnight , and then the cells were treated with PBS, Len-NP, PDT-NP, Combo-NP, PDT-NP+L and Combo-NP+L for 6 h (Lenvatinib and photosensitizer concentration of 5 μM and 2.5 μg/mL).Further incubated with primary CRT antibody for overnight at 4 ℃, and then incubated with the 555-conjugated secondary antibody.Finally, the cells were stained with DAPI, and observed by CLSM.For FCM analysis, cells were seeded in the 12-wells plate at a density of 20 × 10 5 cells/well overnight.Then the cell treatment was the same as CLSM analysis.
Next, the cells were washed with PBS and incubated with CRT antibody.After that, the cells were incubated with secondary antibody for 30 min and then analyzed by FCM.
For evaluating intracellular HMGB1, cover slides were placed into each well of 24-wells plate and cells (1 × 10 5 ) in 1 mL complete media were added.After 12 h incubation, the cells were treated with different formulations like the CRT analysis for 24 h.Next, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, incubated with 1% fetal bovine serum for 30 min.After that, the cells were incubated with primary HMGB1 antibody overnight at 4 ℃, and then incubated with secondary antibody.Finally, the cells were examined by CLSM.
Extracellular secretion of ATP was measured with ATP assay kit according to the manufacture's instruction.The cells were seeded in 12-well plate at a density of 20 × 10 5 cells per well and incubated overnight.Subsequently, the cells were treated with different formulations like the CRT analysis for 24 h.Then the culture medium was collected, 100 μL of ATP detection regents was added into wells and incubated 5 min.Then 20 μL of samples were added to each well and blended quickly.The luminescence of the samples was measured by a microplate reader (SpectraMax M3).

Western bloting
All the samples were washed with clod PBS and lysed with RIPA buffer (Biotech) supplement with PMSF (Biotech) and Phosphatase inhibitor cocktail (Biotech) on ice to extract protein.
The bicinchoninic acid protein assay kit (Beyotime) was used to measure the concentration of protein.Proteins in equivalent amounts were then separated on a 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the PVDF membrane, followed by blocking in TBS-T solution containing 5% skim milk (for phosphorylated proteins) for 1 h.Subsequently, the membrane was incubated with primary antibody against PD-L1 (AF7710, Beyotime) and GAPDH (AF1186, Beyotime) overnight at 4 ℃.Then, second antibodies were added for 1 h at room temperature, and the protein blots were visualized via Xray film.

Animal welfare and protocols
Healthy KM mice (6-8 weeks old), male C57BL/6 mice, and BALB/c nude mice were purchased from SPF Biotechnology (Beijing, China) and raised in SPF animal rooms.All animal experiments reported herein were performed under guidelines evaluated and approved by Peking University Institutional Animal Care and Use Committee (LA2021316)

In vivo biosafety evaluation
Healthy KM mice were randomly divided into 4 groups (n = 3 mice per group).On day 0, 3, 6, and 9, each group mice were injected with PBS, Len-NP, PDT-NP and Combo-NP (photosensitizer concentration of 10 mg/kg, Lenvatinib concentration of 8 mg/kg) through tail vein, respectively.Then the mice were sacrificed at 14 days after treatments, and the blood samples of the mice were collected for hematological and serum biochemical analysis.
Meanwhile, the main organs including heart, liver, spleen, lung and kidney were also obtained for further analysis.All the tissues were paraffin embedded and tissue sections were prepared.
Then H&E staining were performed to observe pathological features, and images were captured using an EVOS XL Core optical microscope (EVOS XL Core, AMG, USA).

Biodistribution of Combo-NP in vivo
OCM1 cells were injected subcutaneously into the left flank of the BALB/c mice.Combo-NP@Cy7.5 was intravenously injected into the mice when the tumor volume reached 200 mm 3 .
An IVIS imaging system was used for fluorescence imaging at 1, 4, 7, 12, 24 and 48 h after injection.Finally, the tumor tissues and major organs (heart, liver, spleen, lung, kidney and intestine) were collected after 48 h injection and quantified using the IVIS Spectrum imaging system.

Antitumor efficacy evaluation in the OCM1 and B16F10 tumor model
To evaluate the antitumor effects of Combo-NP in a human-derived OCM1 tumor model and B16F10 tumor model, the BALB/c nude mice and male C57BL/6 subcutaneous tumor model were constructed by injecting OCM1 cells and B16F10 cells into the left flank of the mice, respectively.When the tumor volume reached approximately 80 mm 3 , the mice were randomly divided into 5 groups (n = 5/group) and PBS, Len-NP, Combo-NP, PDT-NP+L, Combo-NP+L (equivalent to photosensitizer 10 mg/kg, Lenvatinib 8 mg/kg) were injected intravenously via the tail veins.The mice of laser groups were irradiated with a NIR light of 808 nm at intensity of 1 W/cm 2 for 3 min after 24 h injection.All the mice were treated every three days, and the tumor volume and body weight were monitored.The tumor volume was calculated as (length × width 2 )/2.The mice were sacrificed after 15 or 16 days of treatments.

Immunohistochemical and immunofluorescence analyses
After completion of treatments, the mice were sacrificed and then the major organs and tumors were excised for histological observation by H&E staining and immunofluorescence (IF) staining.For H&E staining, excised tumors and organs were fixed in 4% paraformaldehyde solution, embedded in paraffin sections, and stained with hematoxylin and eosin.Sections were then viewed under a fluorescent microscope (IX83, Olympus).TUNEL and Ki67 staining were used to assess apoptosis and proliferation of tumor tissues, respectively.The procedures were consistent with the manufacturer's protocol, and finally detected by CLSM.

Immunogenic cell death in vivo
To evaluate the release of HMGB1 and infiltration of CD8 + T cells in tumor tissues, the mice were sacrificed and tumors tissues were separated, then the tumor tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin.Paraffin tissue sections were stained with primary antibodies overnight at 4 ℃.Secondary antibodies were added and incubated for 1 h.Nuclei were counterstained with DAPI and then the stained sections were imaged with a confocal microscope.

Tumor hypoxia and vascular normalization assays in vivo
To detect tumor hypoxia and tumor vascular normalization.The mice were sacrificed after complete treatments, and tumors tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin.Paraffin tissue sections were stained with anti-Hif-1α to mark tumor hypoxia, followed by processing with second antibodies.Finally analyzed using a confocal microscope.Vascular normalization makers were labeled with anti-CD31 and anti-α-SAM antibodies, firstly, the tumor tissues were fixed, processed and embedded in paraffin, subsequent steps were consistent with the manufacturer's protocol, and finally detected by CLSM.The positive region of CD31 and α-SAM were analyzed by ImageJ.

Systemic antitumor mediated abscopal effect and metastatic tumor models
To evaluate the effects of the combination Combo-NP with α-PD-L1 mAb, B16F10 cells were injected into the left flank (primary tumor) of the C57BL/6 male mice.The mice were randomly grouped (n = 5 /group) including PBS, Len-NP, Combo-NP+L and Combo-NP+L+α-PD-L1 (equivalent to photosensitizer at 10 mg/kg, Lenvatinib at 8 mg/kg and α-PD-L1 at 10 mg/kg) when the tumor volume of primary tumor reached 80 mm 3 .The mice of laser groups were irradiated to primary tumors, while the abscopal tumors were protected from irradiation after 24 h injection.All the mice were treated every three days and α-PD-L1 was injected intraperitoneally on days 1, 4, 7, 10.For the distant primary tumor models, the B16F10 cells were injected into the right flank (abscopal tumor) 4 days after establishment of the primary tumor model.Models of tumor metastasis were constructed by intravenously injecting B16F10 cells into the tail vein 4 days after establishing the primary tumor model.Finally, the mice were sacrificed and lungs were obtained.

Flow cytometry analysis of the animal tissues
The mice were sacrificed after treatments.The obtained fresh tumors, tumor-draining lymph modes and spleens were used to prepare single-cell suspension.Then the suspensions were further incubated with different antibody against the immune cells.To detect the DCs maturation in tumor and lymph modes, the cell suspensions were stained with antibodies of anti-CD11c-PE, anti-CD80-FITC and anti-CD86-APC, respectively.The matured DCs were marked as CD11c + CD80 + CD86 + cells.To detect the infiltration of antitumor T cells, the suspensions of tumor and spleen were incubated with anti-PE-CD3, anti-CD8-FITC and anti-CD4-APC and the CD8 + T cells were denoted as CD3 + CD4 -CD8 + cells.Finally, immunosuppressive cells including MDSC (characterized as CD11b + Gr-1 + ), Tregs (CD4 + Foxp3 + ) and M2 (characterized as F4/80 + CD206 + ) in tumor tissues were also envaulted in the same way as above.

Statistical analysis
Experiments were performed at least three times and results were expressed as means ± SD.
Statistical significances were analyzed using the one-way ANOVA test or two-way ANOVA test.The difference was regarded as significant when the p value was less than or equal to 0.05.

Table S1 .
Antibodies used for flow cytometry analysis